In the United States in 2002, over 20 million people had diabetes, which contributed to over 224,000 deaths and the total cost of diabetes was $132 billion. A worldwide epidemic of diabetes is now projected which will have serious healthcare and economic consequences. The discovery of new biomarkers will be very important for much-needed advances in screening, diagnosis, prognosis, prediction of disease recurrence and therapeutic monitoring for these devastating diseases. The goal for Phase I of this Project is to optimize in human plasma a new proteomics technology, Differential Capture Proteomics (DCP), which can identify the differences in protein composition between two biological samples. This technology also simultaneously creates affinity reagents for each of the identified difference proteins. A patent for DCP has been issued to Differential Proteomics Inc. The goal for a subsequent Phase II study is to apply DCP, newly optimized for human plasma, to the discovery of biomarkers of diabetes using appropriate patient plasma samples to enable novel protein isolation and identification, novel affinity reagent creation, assay formulation, and clinical validation. Studies in our laboratory to optimize the DCP technology have successfully focused on a model system. However, the capabilities of DCP for diabetes biomarker discovery in patient plasma samples have yet to be demonstrated with an optimization study. Such a study is proposed here, in which doped diabetes biomarkers (representing a model diabetes patient sample) will be tested for detectability at clinical sample level concentrations. The proposed study will use five specific diabetes protein antigens, for which there are commercially available immunoassays. The success of this Phase I study will lead to a Phase II program for testing multiple diabetes patient plasma samples for novel biomarker discovery, assay generation, and validation. Specific Aim 1: Confirm that three pooled Random Peptide Phage Libraries actually contain binding phage species against each of the five diabetes antigens chosen. Specific Aim 2: Show how well Differential Capture Proteomics detects the differences between two human plasma samples, one of which is doped with the mixture of five diabetes antigens. Specific Aim 3: Establish the specificity of the affinity capture reagents that are generated as part of the process.